47th Annual Meeting and Exposition
December 10-13, 2005

Georgia World Congress Center
Atlanta, Georgia

Scientific Program

Please note that duplication and recording are prohibited in the session rooms.

The 2005 Scientific Committee sessions will be held Saturday, December 10, and Sunday, December 11. Each session will be offered twice unless otherwise noted. Invited abstracts of these sessions will be published in the November 16 abstract issue of Blood (the Program and Abstracts Book) and the Annual Meeting Notebook.

Gene expression profiling promises to deliver a quantitative and reproducible molecular diagnosis of human lymphomas. We have created statistical algorithms based on gene expression analysis of pretreatment biopsy samples that can diagnose most common lymphoma subtypes with 100% accuracy and distinguish benign conditions. In addition, we have used gene expression profiles to define subgroups of diffuse large B-cell lymphoma that are histologically indistinguishable but that differ with respect to their oncogenic abnormalities and clinical outcomes. Moreover, these subgroups utilize different signaling pathways for their proliferation and survival, thus providing new rational targets for therapy. In diffuse large B-cell lymphoma, mantle cell lymphoma, and follicular lymphoma we have developed gene expression-based models of survival that stratify patients into strikingly disparate risk groups. Importantly, these predictive models identify biological features of the tumors that influence their clinical behavior, including the tumor proliferation rate and the character of tumor-infiltrating immune cells. A single DNA microarray comprised of several hundred genes is capable of delivering both molecular diagnosis and molecular prognosis for all human lymphomas, allowing us to envision a future in which every lymphoma patient receives a diagnostic gene expression profile.

Carlo M. Croce, MD, The Ohio State University Comprehensive Cancer Center, Columbus, OH
MicroRNAs and Microarrays in Lymphoma/Leukemia

MicroRNA expression profiles can distinguish normal B cells from malignant B cells in chronic lymphocytic leukemia (CLL). We investigated whether microRNA profiles are associated with known prognostic factors in CLL, and we evaluated the microRNA expression profiles of 94 samples of CLL cells for which ZAP-70 expression, mutations in the rearranged IgV_H gene, and the time from diagnosis to initial treatment were known. We also investigated the presence of abnormalities in the genomic sequence of 42 microRNA genes. A unique microRNA expression signature composed of 13 genes (of 190 analyzed) can differentiate cases with low or high ZAP-70 expression and cases with unmutated or mutated IgV_H. The same microRNA signature was associated with the presence or absence of disease progression. We also identified a germline mutation in miR-16-1/miR-15a primary precursor. This mutation caused low levels of microRNA expression in vitro and in vivo and was associated with deletion of the normal allele. Germline or somatic mutations were found in 5 out of 42 sequenced microRNAs in 11/75 CLL patients, but none of these were found in 160 subjects without cancer (p<0.0001). Thus, a unique microRNA signature is associated with prognostic factors and disease progression in CLL. Mutations in microRNA transcripts are frequent and may have functional importance.

Thomas J. Kipps, MD, PHD University of California – San Diego, La Jolla, CA
Zap70 in Chronic Lymphocytic Leukemia: Biology, Diagnosis, and Prognostic Importance

Patients with chronic lymphocytic leukemia (CLL) cells that use unmutated immunoglobulin (Ig) variable (V) genes typically have a more aggressive disease than patients with CLL cells that use Ig V genes with somatic mutations. Microarray analyses, however, revealed that only a relatively small subset of genes were differentially expressed and could be used to distinguish these two CLL subgroups. One of these encodes ZAP-70, a 70-kDa-protein tyrosine kinase initially found in T cells. In contrast to CLL cells with mutated Ig V genes, CLL cells with unmutated Ig V genes generally express ZAP-70. However, this association is not absolute and, in fact, ZAP-70 may be a stronger predictor than Ig V gene mutational status of the need for early treatment. This suggests that the expression of ZAP-70 per se is more closely associated with the mechanism(s) governing the more aggressive nature of CLL with unmutated Ig V genes. Ordinarily, ZAP-70 plays a critical role in T-cell receptor signaling. Similarly, ZAP-70 contributes to effective signaling of the CLL surface Ig receptor, which typically is not highly functional in CLL cells lacking ZAP-70. Moreover, gene transfer of ZAP-70 into formerly ZAP-70-negative CLL cells can improve Ig receptor signaling. These results indicate that ZAP-70 enhances Ig receptor signaling, which, in view of the remarkable restriction in the Ig expressed in CLL, probably provides a growth and/or survival stimulus for leukemia B cells. Consistent with this notion, ZAP-70+ CLL cells appear more activated than ZAP-70-negative CLL cells. Recent studies found that ZAP-70+ CLL cells, but not ZAP-70+ T cells, distinctively express activated heat-shock protein 90 (Hsp90), which forms a multi-protein chaperone complex that binds and stabilizes ZAP-70. Consequently, drugs that can inhibit activated Hsp90, such as 17-allyl-amino-demethoxy-geldanamycin (17-AAG), can cause selective degradation of ZAP-70, loss of effective Ig-signaling, and apoptosis of ZAP-70+ CLL cells at concentrations that have no apparent effect on normal T cells or ZAP-70-negative CLL cells. As such, CLL-cell expression of ZAP-70, an adverse prognosis marker, may actually become the Achilles heal of this CLL subtype, allowing for effective targeted-therapy of aggressive disease.

Myeloproliferative disorders (MPD) are characterized by enhanced proliferation and survival of myeloid lineage hematopoietic progenitors due to acquired somatic mutation. The first genetic insights into disease pathogenesis, and the subsequent development of molecularly targeted therapies for MPD, came from cloning of chromosomal translocation breakpoints. Examples include the BCR-ABL gene rearrangement associated with chronic myelogenous leukemia (CML), and a family of PDGFR gene rearrangements associated with chronic myelomonocytic leukemia (CMML). Each of these are constitutively activated tyrosine kinases that recapitulate the human phenotype in murine models of disease, and are sensitive to inhibition by imatinib in vitro and in vivo. Following in this same vein, systemic mast cell disease (SMCD) has been shown to be associated with activating mutations in KIT, and hypereosinophilic syndrome (HES) was shown to be associated with the FIP1L1-PDGFRA fusio tyrosine kinases by virtue of clinical response to empiric imatinib therapy. However, until recently, the genetic basis of the largest collective group of MPDs - polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis with myeloid metaplasia (MF), was not known. Several groups have reported that a single mutant allele, JAK2V617F, accounts for the majority of cases of PV, ET, and MF. JAK2V617F is a constitutively activated tyrosine kinase that confers factor independent growth to murine hematopoietic cell lines expressing the erythropoietin receptor, and is expressed in the HEL cell line. JAK2V617F is sensitive to inhibition with selective small molecule inhibitors and is an attractive target for therapeutic intervention. Data will be presented that begins to address several of the important outstanding questions related to the role of JAK2V617F in these MPD, including the basis for phenotypic disparities among patients with a single known disease allele; why there is only one known activating allele of JAK2 among all patients with these MPD; the effects of expressing the mutant JAK2 allele in murine models of disease; and the genetic basis for disease in PV, ET, and MF patients that lack the JAK2V617F mutation.

Kenneth Kaushansky, MD, University of California – San Diego, San Diego, CA
Thrombopoietin (TPO) and Its Receptor, c-Mpl: Expected and Unexpected Contributions to Normal and Malignant Hematopoiesis

Hematopoiesis is a complex process in which both cell intrinsic (primarily transcription factors) and exogenous (predominantly hematopoietic cytokines) factors promote the survival, proliferation, and differentiation of hematopoietic stem cells (HSC) into the 4 x 10 ¹¹ mature blood cells produced daily. While widely recognized as the primary regulator of thrombopoiesis, TPO has proven to exert important and non-redundant actions on HSCs. For example, genetic elimination of Tpo or its receptor in mice reduces the steady-state number of transplantable HSCs ~10-fold, and the expansion of HSCs following transplantation by nearly 20-fold. In humans, homozygous or compound heterozygous, severe missense or non-sense mutation of the c-Mpl receptor causes congenital amegakaryocytic thrombocytopenia in children, a disorder that nearly always eventuates in stem cell exhaustion and aplastic anemia. The molecular bases for many of these actions are becoming increasingly understood. While known to be critical for embryonic body pattern development for decades, the Hox genes have more recently been found to contribute to adult biology, including hematopoiesis. We have shown that HoxB4 and HoxA9 are responsible, in part, for the favorable effects on TPO on HSCs, as HoxB4 transcription is stimulated by TPO in a p38 MAPK and USF1/2 dependent manner, and the nuclear localization of HoxA9 is regulated by TPO through its effects on the HoxA9 partner protein, MEIS1. The autocrine production of cytokines plays several roles in the inflammatory response, and more recently, the autocrine production of vascular endothelial cell growth factor (VEGF) has also been shown to be essential for HSC maintenance. We have shown that TPO affects stem cell VEGF production by affecting the stability of the primary factor for VEGF transcription, hypoxia inducible factor (HIF) 1α. Together, these TPO-dependent mechanisms promote stem cell self renewal and expansion, and imply that a number of apparently cell-intrinsic mechanisms of HSC homeostasis (i.e., transcription factors) can be profoundly influenced by cell-extrinsic processes.

Ivo Touw, PhD, Erasmus University Medical Center, Rotterdam, The Netherlands
Perturbed G-CSF Receptor Functions in Severe Congenital Neutropenia, MDS, and AML

G-CSF, the major growth factor involved in neutrophil development, activates its cognate receptor (G-CSF-R) through induction of homo-oligomeric receptor complexes. Signaling substrates involved in G-CSF-induced proliferation and survival include STAT5 and PKB, activated via the juxtamembrane region of G-CSF-R, and p21 Ras and Erk1/2, activated via one of the four conserved tyrosine residues (Y764) in the G-CSF-R C-terminus. G-CSF-R is unique in its ability to initially transduce proliferation and survival signals in myeloid progenitors, followed by signals that drive growth arrest and terminal myeloid differentiation. This bimodal response depends on multiple mechanisms, involving distinct regions in the cytoplasmic domain of G-CSF-R. For instance, STAT3 and SOCS3, implicated in G-CSF-induced growth arrest, are recruited via Y704/Y744 and Y729, respectively. Another mechanism playing a major role in balancing G-CSF signals involves the kinetics of G-CSF-R internalization (controlled by a dileucine-based internalization motif) and routing (involving conserved lysine residues that are target for E3-mediated ubiquitylation). Finally, the most distal part of G-CSF-R (a.a. 792-813), comprising a WD40-repeat interaction domain that binds the SOCS box-containing proteins Wsb1 and 2, negatively controls proliferation. We are currently exploring a model in which constitutive forward routing of G-CSF-R is controlled by the Wsb proteins and ligand-induced downward (lysosomal) routing by SOCS3. Several mutations and functional polymorphisms in the G-CSF-R have been identified in AML, myelodysplasia (MDS), and severe congenital neutropenia (SCN). Most frequently, mutations are detected in SCN patients who receive G-CSF treatment. These acquired nonsense mutations, truncating the G-CSF-R at positions between amino acid 715 and 732, are strongly associated with (but not always heralding) malignant transformation of SCN. The C-terminal truncations lead to the loss of SOCS3 and Wsb recruitment and give rise to constitutive membrane localization, resulting in strongly increased proliferative signals and prolonged activation of STAT5. While this provides a plausible explanation for the hyperproliferation and clonal expansion of progenitor cells harboring these mutations, it is remains unclear how this links to malignant transformation to MDS/AML. Interestingly, recent studies from the Corey lab in the gcsfr∆715 knock-in mouse model show that G-CSF induces excessive production of reactive oxygen species (ROS) in gcsfr∆715 bone marrow cells, which might contribute to leukemic progression. G-CSF-R mutations are only rarely found in de novo AML and MDS. However, it was recently reported that a rare single-nucleotide polymorphism (G-CSF-R_Glu785Lys) predisposes individuals to high-risk MDS.¹ This polymorphic variant of G-CSF-R has reduced proliferative signaling abilities, presumably due to abnormal routing caused by the additional lysine. How this associates with predisposition to high-risk MDS remains to be clarified.

Erythropoietin (EPO), a member of the growth hormone/prolactin cytokine family, was first characterized as a hematopoietic growth factor produced in adults by the kidneys, which increases red blood cell production by inhibiting apoptosis in early RBC. EPO has been used by millions of patients over the last decade for the treatment of anemia. Like other members of its cytokine superfamily, EPO and its receptor have been found to be expressed by other tissues including the central nervous system (CNS). The up-regulation of EPO and its receptors in the brain by hypoxia/ischemia in vitro and in vivo suggest that this cytokine is an important mediator of the brain’s response to injury. Originally thought to be an independent cytokine system, we have recently shown that peripherally administered EPO is specifically carried across the blood-brain, -retina, -spinal cord, and -peripheral nerve barriers, where it exhibits tissue protection. However, in non-anemic patients, native EPO can cause polycythemia with prolonged use. We have recently developed derivatives, e.g., carbamylated EPO “CEPO” that are not erythropoietic but retain tissue protective activities in vivo. Animal studies conducted to date support the use of CEPO derivatives in such disease models as retinal ischemia, myocardial infarction, stroke, brain and spinal trauma, ALS, epilepsy, and multiple sclerosis. The molecular basis for the selectivity of CEPO having tissue protective but not erythropoietic activities derives from the existence of distinct receptors. Erythropoiesis is signaled through the well-known homodimer of EPO receptor subunits, while tissue protection signals through a heteromeric receptor that comprises the EPO receptor subunit and a beta common receptor subunit. Selective signaling of the latter receptor complex provides the rationale for safely administering tissue protective compounds in diverse diseases, especially those requiring chronic therapy, as well as facilitating the identification of more potent derivatives and small molecule agonists.

Soren K. Moestrup, MD, PhD, University of Aarhus, Aarhus, Denmark
Vitamin B12 Absorption by Means of the Cubilin/Amnionless Receptor System

Vitamin B12/cyanocobalamin (B12) is a tetrapyrrol structure like heme. The cellular uptake of B12 bears resemblance to heme and hemoglobin uptake, which rely on specific carrier proteins and endocytic receptors. The carrier/receptor system for B12 for uptake in the intestine is accounted for by at least three different proteins. In the intestine, B12 binds avidly to gastric intrinsic factor (IF) and the IF-B12 is subsequently recognized and endocytosed by a large receptor complex formed by the products of two genes, cubilin and amnionless (AMN), expressed in the epithelium of the terminal ileum. Cubilin is a 460 kDa peripheral membrane protein containing a high-affinity binding site for IF-B12 and a binding site for the 45 kDa AMN protein. AMN is an integral membrane protein with a transmembrane segment and a cytoplasmic tail containing endocytic signals. The cubilin/AMN receptor complex is a structure that is strikingly different from other known endocytic receptors. Additionally, cubilin/AMN is expressed in the polarized epithelium of the kidney and other tissues where the cubilin unit binds a broad spectrum of other protein ligands. Biochemical studies showing cubilin/AMN as a functional receptor complex are supported by genetic analyses of inborn B12 deficiency (Imerslunds-Gräsbeck syndrome /megaloblastic anemia-1). In agreement with the coordinated function of both receptor units, disease-causing mutations in AMN and cubilin genes cause the same disease phenotype characterized by B12 malabsorbtion and B12-deficiency symptoms such as megaloblastic anemia and neurological/mental problems. Furthermore, the vitamin deficiency is often associated with proteinuria, in accordance with an additional role of cubilin/AMN in renal protein reabsorption. Studies are now in progress to further characterize the structure of the complex and the intracellular assembly and trafficking of cubilin and AMN to the membrane. These studies are, to a large extent, carried out by functional and morphological investigations of cultured cells transfected with mutant receptor units. Additionally, a thoroughly characterized dog model of Imerslund-Gräsbeck syndrome continues to have an important role in elucidating the function of the cubilin/AMN complex in vivo. This model has been more useful than rodent models so far because cubilin and AMN have additional but not yet defined roles essential for rodent embryonic development.

Co-Authors: Mette Madsen, PhD, University of Aarhus, Aarhus, Denmark; and Qianchuan He, PhD and John C. Fyfe, PhD, College of Veterinary Medicine, Michigan State University, East Lansing, MI

David A. Williams, MD, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH
Rac’n and Rho’llin: GTPases in Blood Cell Development and Function

Rho GTPases regulate multiple cell functions, including cell shape, migration and adhesion, proliferation, and survival. We have reported the key roles that the related Rho GTPases Rac1 and Rac2 play in hematopoiesis¹ and specific roles these GTPase play in neutrophil biology^2,3. Indeed, mutations in Rac2 have been implicated in a human phagocytic immunodeficiency disease⁴. We have more recently examined the role of the Rho GTPases Rac1 and Rac2 in HSC engraftment and mobilization⁵. Rac1, but not the hematopoietic-specific Rac2, is required for the engraftment phase of hematopoietic reconstitution, since Rac1^-/- HSC fail to rescue in vivo hematopoiesis after transplantation, but induced deletion of Rac1 sequences after initial engraftment does not impair steady-state hematopoiesis. Rac1^-/- HSC/P show impaired spatial localization to the endosteum but near normal homing to the medullary cavity in vivo. Interaction with the bone marrow microenvironment in vitro is markedly altered. While post-engraftment loss of Rac1 alone does not impair hematopoiesis, combined loss of Rac1 and Rac2 leads to ineffective hematopoiesis-associated massive mobilization of HSC from the marrow and intense selection for Rac-expressing HSC. This mobilization is reversible by re-expression of Rac1. In addition, a rationally designed, small molecule inhibitor of Rac activation⁶ leads to transient mobilization of engraftable HSC/P. Rac proteins thus differentially regulate engraftment and mobilization phenotypes suggesting that these biological processes and steady-state hematopoiesis are biochemically separable and that Rac proteins may be important molecular targets for stem cell modification. We also have examined the role of these GTPases in erythropoiesis. The peripheral blood of gene-targeted mice deficient in Rac1 and Rac2 demonstrate a microcytic anemia characterized by severe pyropoikliocytosis, cell fragmentation, and reticulocytosis. Preliminary data suggest that the red cell ghosts of these animals have altered actin: spectrin ratio and an increase in soluble actin. Combined Rac1 and Rac2 deficiency, but not Rac1 or Rac2 deficiency alone, resulted in irregular spectrin heptagon meshwork and abnormal actin aggregation on the red cell surface. Thus, Rho GTPases appear to be key regulators of multiple hematopoietic cell functions, and in erythrocytes Rac signaling appears to be critical for maintenance of red cell membrane integrity by dynamic control of actin interactions with spectrin.

Co-Authors: Theodosia Kalfa, MD, PhD, and Yi Zheng, PhD, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH

References:

1. Gu Y, Filippi MD, Cancelas JA, Siefring JE, Williams EP, Jasti AC, Harris CE, Lee AW, Prabhakar R, Atkinson SJ, Kwiatkowski DJ, Williams DA. Hematopoietic cell regulation by Rac1 and Rac2 guanosine triphosphatases. Science 302:445-449, 2003.

2. Roberts A, Kim C, Zhen L, Lowe J, Kapur R, Petryniak B, Spaetti A, Pollock J, Borneo J, Bradford GB, Atkinson SJ, Dinauer MC, Williams DA. Deficiency of the hematopoietic cell-specific Rho-family GTPase, Rac2, is characterized by multiple abnormalities in neutrophil function and impaired host defense. Immunity 10:183-196, 1999.

3. Filippi MD, Harris CE, Meller J, Gu Y, Zheng Y, Williams D. Unique intracellular localization of Rac2 specifies superoxide generation, actin polarity and chemotaxis in neutrophils. Nature Immunology 5:744-751, 2004.

4. Williams DA, Tao W, Yang F, Kim C, Gu Y, Mansfield P, Levine JE, Petryniak B, Derrow CW, Harris C, Jia B, Ambruso D, Lowe J, Atkinson SJ, Dinauer MC, Boxer L. A dominant negative mutation of the hematopoietic-specific RhoGTPase, Rac2, is associated with a human phagocyte immunodeficiency. Blood 96(5):1646-1654, 2000.

5. Cancelas J, Prabhakar R, Lee A, Zheng Y, Williams, DA. Rac1 and Rac2 Rho GTPases distinctly regulate bone marrow hematopoietic stem cell localization and are novel targets for stem and progenitor mobilization. Nature Medicine 11(8):886-891, 2005.

6. Gao Y, Dickerson J, Guo B, Zheng F, Zheng Y. Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci U S A 101(20):7618-7623, 2004.

For many years, it was widely held that thrombogenesis ensued when a physical barrier separating Tissue Factor (TF) from the procoagulants in the circulation was breached. That is, thrombosis was initiated by physical damage to the vascular wall, thereby enabling the interaction of TF with factor VII/VIIa, thus forming the catalytic complex that enables the initiation of coagulation. TF is known to exist in three forms: the originally discovered full length TF, a shorter, soluble TF created for laboratory use (sTF), and an alternatively spliced TF. The full-length species consisting of 263 amino acids contains a classical transmembrane domain, a short cytoplasmic domain, and the extracellular domain that binds (at least) factors VII and X. The extracellular domain, which shares a common sequence with sTF, exhibits considerable lipid-dependent coagulant activity, and has not been identified in the circulation, whereas an alternatively spliced form that has minimal activity commonly circulates in relative abundance. The only form in which functional, circulating TF is found is the full-length species, most likely inserted into microparticles. These particles apparently are prothrombotic because they have lost their membrane asymmetry and display phosphatidylserine on their surface. We have published experiments in which native whole blood was perfused over collagen-coated glass or porcine arterial tissue; in each instance, abundant TF-positive thrombi formed, containing platelets and fibrin. Addition of a potent TF-blocking agent, factor VIIai, prevented thrombus formation and fibrin deposition. Furthermore, in vivo experiments showed TF-positive material within venous thrombi. The addition of a monoclonal anti-TF antibody blocked further thrombus growth (i.e., fibrin accretion), thereby indicating the functional role of TF in in vivo thrombus propagation. Other laboratories have contributed transgenic mouse models that showed the participation of blood-borne TF in in vivo thrombogenesis. We note that this evidence of circulating active (or activatable) TF relates to its role in thrombogenesis and not necessarily to its ability to shorten whole blood clotting time. Our hypothesis is that when sheared over an appropriate surface, TF is deposited within nascent thrombi, whereupon its activity becomes manifest. We note that the entire process likely starts with a vascular injury, exposing wall-bound TF and that sTF is mainly involved in thrombus propagation.

Co-Author: James J. Hathcock, PhD, Mount Sinai School of Medicine, New York, NY

Kenneth G. Mann, PhD, University of Vermont, Burlington, VT
The Role of Tissue Factor (TF) in Blood Clotting in the Healthy

In the traditional view of blood coagulation, TF expressed by extravascular tissue binds plasma FVIIa and triggers coagulation. Observations of TF antigen in blood have led to the hypothesis that blood TF is the initiator of coagulation and an essential contributor to the maintenance of thrombin generation. We have studied these issues using functional and immunoassays for TF and in vitro reconstructions using a) computer models; b) reconstitutions of the coagulation proteome; and c) corntrypsin-inhibited whole blood. Monoclonal immunoassays for TF reveal small quantities (≤2 pM) of TF-related antigen in plasma. Functional assays of blood from healthy individuals reveal less than 20 fM TF activity. The proposed contribution of blood TF to maintain a coagulation reaction was explored with computer simulations in which TF was eliminated at various points during the reaction. When started with 5 pM, the TF reaction initiation phase (IP) is approximately 300 seconds. During the IP, FXa generation is progressively assumed by the FVIIIa-FIXa complex, and the contribution of TF at various points during the IP becomes progressively less as the propagation phase (PP) is approached. At the inception of the latter, elimination of TF activity has no effect. These constructs were replicated in synthetic plasma proteome models and with whole blood activated with 5 pM TF by quenching the TF-FVIIa complex with inhibitor antibodies to fVIIa and TF. The data are consistent with a TF requirement for starting the reaction and no requirement during the PP of thrombin generation. The need for continuous TF addition to maintain thrombin generation was explored using simulations for additions of fresh charges of TF free “plasma” reactants following the initial quelling of thrombin generation. Subsequent additions result in immediate bursts of thrombin generation indicating that sufficient catalysts already present in the reaction system will supply vigorous thrombin generation independent of the addition of more TF. These results were confirmed empirically with the synthetic plasma proteome system. In whole blood when the reaction reaches a plateau of thrombin-antithrombin (TAT) generation and no more thrombin is being generated, an added fresh charge of blood results in a new burst of TAT. These data indicate that 1) TF from blood is not required when an exogenous source is present; 2) the reaction once past the IP no longer requires TF; and 3) apparently quiescent reactions have the ability to generate additional amounts of thrombin when a fresh charge of blood is admitted to a wound site. These data indicate that at the site of a vascular injury clotting is initiated by local TF expression and will continue until blood flow into the wound area has ceased.

Co-Authors: Thomas Orfeo, PhD, and Behnaz Parhami-Seren, PhD, University of Vermont, Burlington, VT

Nigel S. Key, MD, University of Minnesota Medical School, Minneapolis, MN
Microparticle Metrics, with Special Emphasis on Tissue Factor

Circulating peripheral blood microparticles (MPs) have been described by many laboratories. Enumeration and/or characterization of MPs may provide insights into the extent of activation or apoptosis of the cells from which they arise, thereby presenting potential diagnostic opportunities (e.g. for platelet-derived MPs in the diagnosis of HIT). In addition, MPs may possess intrinsic effector functions, participating in inter-cellular communications in inflammation, vascular reactivity, and angiogenesis. MP-dependent (tissue factor (TF) and prothrombinase) procoagulant activities may also contribute to normal hemostasis and pathological thrombosis. While platelets are the dominant source of MPs in health, other cells, including endothelium, monocytes, granulocytes, and erythrocytes, contribute to the circulating pool in disease states. There is no consensus regarding the minimal criteria that define a circulating MP, nor for the methods that should be employed for their detection. Most groups have defined circulating MPs according to at least two criteria: 1] size below a pre-defined threshold – most commonly < 1.0µm, and 2] specific binding of monoclonal antibodies to cell-specific antigens. MPs should be distinguished from other sub-cellular particles including apoptotic bodies, organelles, and exosomes. Phosphatidylserine (PS) exposure on MPs (detected by annexin V binding) is an additional criterion used by many groups to define MPs. This more restrictive definition may overlook a potentially important MP subset, namely those that do not express membrane PS. MPs that do not bind annexin V in vitro may be induced to expose anionic phospholipids – thereby gaining full procoagulant activity – with a second activation step. Most of the evidence for TF expression by MPs has employed flow cytometry methods using monoclonal Abs to detect TF antigen. TF procoagulant activity may be measured on isolated or captured MPs obtained by ultracentrifugation or MoAb capture, respectively. Here again, the reference material and method for the assay of TF activity have not been defined. TF ‘encryption’ refers to the phenomenon that membrane-bound TF expresses only a fraction of its full procoagulant potential in the resting compared to the disrupted cell. MP-borne TF activity (i.e., the state of TF ‘encryption’) also depends on the membrane phospholipid composition. It is conceivable that MPs bearing ‘encrypted’ TF are primarily responsible for shuttling the TF molecule between cells, whereas MPs bearing ‘de-encrypted’ TF PCA directly participate in thrombin generation.

Iron is a required element for all eukaryotes but is toxic in high concentrations. Consequently, iron acquisition is tightly coordinated with iron utilization. Studies in the budding yeast Saccharomyces cerevisiae have shown how iron transport across the plasma membrane is regulated by mitochondrial biosynthetic activities. Mitochondria house two processes that utilize iron, heme synthesis, and iron sulfur cluster synthesis. Most enzymes that utilize the redox active metals iron and copper are involved in respiratory activities, oxygen binding, and detoxification of oxygen. Heme formation is sensitive to oxygen tension since porphyrin synthesis requires oxygen. Decreased heme synthesis leads to a decrease in the transcription of transport systems for the iron and copper but not for the redox inactive transition metal zinc. The lack of transcription for the iron and copper regulons is a consequence of a transcriptional repressor, which is relieved by heme. Thus, in the absence of oxygen or heme, there is decreased expression of metallo-proteins that are no longer useful, as well as reduced accumulation of potentially dangerous metals. In yeast, the synthesis of iron-sulfur clusters is restricted to the mitochondria where this prosthetic group is synthesized and exported to the cytosol. Decreased mitochondrial iron sulfur cluster synthesis leads to increased cellular iron accumulation due to increased transcription of genes that encode iron transporters. Mutations in genes that encode enzymes involved in iron-sulfur cluster synthesis lead to excessive mitochondrial iron accumulation. Proteins involved in iron-sulfur cluster synthesis are highly conserved and excessive mitochondrial iron accumulation is seen in humans with mutations in genes that affect iron-sulfur cluster synthesis, such as frataxin (Friedreich Ataxia) and ABC7 (Sideroblastic Anemia with Ataxia). These results indicate that in humans as in yeast, mitochondrial metabolism affects iron homeostasis.

Mario Cazzola, MD, University of Pavia Medical School, Pavia, Italy
Molecular Basis of Sideroblastic Anemias

The sideroblastic anemias are a heterogeneous group of inherited and acquired disorders characterized by anemia of varying severity and the presence of ring sideroblasts in the bone marrow. These latter are immature red cells with iron-loaded mitochondria visualized by Prussian blue staining as a perinuclear ring of blue granules. The most common of the inherited forms is X-linked sideroblastic anemia (XLSA, OMIM 301300), which is caused by mutations in the erythroid-specific ALA synthase gene (ALAS2). The most common acquired sideroblastic anemia is a myelodysplastic syndrome (MDS) defined as refractory anemia with ring sideroblasts (RARS). The nature of the iron deposited in iron-loaded mitochondria of ring sideroblasts has been clarified recently. Mitochondrial ferritin (MtF) is encoded by an intronless gene on chromosome 5q23.1; most of the iron deposited in perinuclear mitochondria of ring sideroblasts is present in the form of MtF. In patients with XLSA, defective ALAS2 enzyme activity leads to insufficient protoporphyrin IX synthesis, mitochondrial iron accumulation, and apoptosis of red cell precursors. Overexpression of MtF likely results from defective iron utilization and partly protects mitochondria from iron-induced damage. However, the expanded erythropoiesis suppresses hepcidin production leading to excessive iron absorption and reticuloendothelial iron release. Symptoms of hemochromatosis can be the first manifestation of disease in middle-aged men, and coinheritance of the HFE C282Y mutation worsens parenchymal iron overload. Most patients with XLSA are to some extent responsive to pyridoxine; iron overload may suppress this responsiveness, and its reversal by phlebotomy generally results in higher hemoglobin levels during pyridoxine supplementation. The molecular basis of RARS is less understood. Using flow cytometry immunophenotyping, we analyzed the expression of CD71 (transferrin receptor), cytosolic H- and L-ferritin, and MtF in immature red cells from MDS patients. Compared with controls, myelodysplastic erythroid cells have lower expression of CD71 and higher expression of cytosolic ferritins, i.e., an iron-loaded phenotype . MtF is specifically detected in patients with ring sideroblasts and is expressed at a very early stage of erythroid differentiation, at a time when significant mitochondrial iron accumulation is unlikely. Interestingly, immunophenotypic analysis shows higher levels of CD71 expression in MtF-positive than in MtF-negative erythroid cells; this may reflect a cytosolic iron deprivation induced by MtF.

Jane-Jane Chen, PhD, Massachusetts Institute of Technology, Cambridge, MA
Heme-Regulated Translation in Erythroid Differentiation and Disease

During erythroid differentiation and maturation, it is critical that the three components of hemoglobin, α-globin, β-globin, and heme, are made in proper stochiometry to form stable hemoglobin. Heme-regulated translation mediated by the heme-regulated eIF2α kinase (HRI) provides one of the mechanisms that ensure the balanced synthesis of globins and heme. HRI is a heme-regulated protein kinase that phosphorylates the a -subunit of eukaryotic translational initiation factor 2, thereby inhibiting protein synthesis globally. It is expressed predominantly in erythroid cells and is activated in iron/heme deficiency. Thus, HRI serves as a feedback inhibitor of globin synthesis by sensing the intracellular concentration of heme. Heme binds directly to the two unique domains of HRI and prevents its activation by autophosphorylation. HRI is essential for the translational regulation of globins and the survival of erythroid precursors in iron deficiency. It is responsible for the physiological adaptation to the microcytic, hypochromic anemia in iron-deficiency. In the absence of HRI, both α and βglobins are synthesized in amounts greater than the heme available. These heme-free globins are unstable and precipitate in red cell precursors, resulting in a more deleterious hyperchromic, normocytic anemia with accelerated apoptosis of erythroid precursors. The protective function of HRI has also been demonstrated in two murine models of human intrinsic red cell disorders, erythropoietic protoporphyria and β-thalassemia. In both cases, the presence of HRI reduces the severity of these diseases, and the maximal beneficial effect is achieved when both copies of the HRI gene are retained, as HRI haploinsufficiency result in more severe clinical and pathological manifestations. Thus, HRI may be one of the modifiers that influence the outcome of erythropoietic protoporphyria and β-thalassemia in humans. Furthermore, translational regulation by HRI is important in safeguarding the level of the globin protein production under various conditions. It is critical to reduce excess synthesis of globin proteins or heme under non-optimal stress conditions or disease states. The role of HRI may be broader and more general in other intrinsic red cell disorders since HRI is also activated by a number of cytoplasmic stresses other than heme deficiency. To date, no disease in human or mouse is known to be associated with mutations in the HRI gene. Identification of HRI mutations in human red cell disorders will be an important area of future investigation. Additionally, the prospect of HRI and its downstream substrates as potential pharmaceutical targets for treatment of red cell disorders would be worthy of investigation.

B-cell derived non-hodgkin lymphoma (B-NHL) represent a heterogeneous group of malignancies that, with the exception of mantle cell lymphoma, arise by malignant transformation of B cells within the germinal center (GC). Within GC, antigen-stimulated B cells undergo rapid proliferation and specific genome remodeling processes such as immunoglobulin (Ig) somatic hypermutation (SHM) and class switch recombination (CSR). The pathogenesis of B-NHL is associated with the alteration of various proto-oncogenes and tumor suppressor genes by mechanisms common to other cancer types, such as gene amplification and deletion, as well as by two mechanisms that involve errors in genetic functions specific to GC B cells: i) chromosomal translocations that lead to deregulated expression of oncogenes (BCL2, BCL6, c-MYC) and are thought to derive from DNA breaks associated with the Ig remodeling mechanisms (VDJ recombination, SHM, and CSR); and ii) aberrant somatic hypermutation (ASHM), which causes mutations in the 5' region of multiple oncogenes and is due to the misfiring of SHM on non-physiologic targets¹; recent studies have identified an increased number of ASHM targets and shown that some of the ASHM-derived mutations lead to the oncogenic activation of target genes including c-MYC. A common pathogenetic target of both translocations and SHM and a required regulator of GC development is the BCL6 proto-oncogene, which encodes a transcriptional repressor necessary for GC formation. Deregulated expression of the BCL6 gene caused by chromosomal translocation or SHM of its 5’ regulatory region² is common in DLBCL (~40% of cases) and leads to DLBCL development in transgenic mice³. One major function of BCL6 is to repress GC B cell responses to genotoxic stress via direct suppression of p53 transcription or via MIZ1-mediated suppression of the cell-cycle regulator p21^4,5; these activities are thought to allow the rapid proliferative expansion of GC as well as the execution of the physiologic genomic break/recombination events required for CSR and SHM. Notably, recent studies have identified a signaling pathway that down-regulates BCL6 expression in response to increasing levels of DNA damage and suggest that the ability of GC B cells to sustain genotoxic-stress is regulated, via BCL6, by the level of DNA damage itself. These results imply that B-NHL that constitutively express BCL6 may be functionally impaired in apoptotic and DNA-damage responses and that therapeutic targeting of BCL6 may represent an attractive strategy to inactivate the oncogene as well as to restore normal genotoxic responses.

References:

1. Pasqualucci L, Neumeister P, Goossens T, Chaganti RSK, Küppers R, Dalla-Favera R. Hypermutation of multiple proto-oncogenes in B-cell diffuse large cell lymphoma. Nature 2001;19:341-346.

2. Pasqualucci L, Migliazza A, Basso K, Houldsworth J, Chaganti RSK, Dalla-Favera R. Mutations of the BCL6 proto-oncogene disrupt its negative autoregulation in diffuse large B-cell lymphoma. Blood 2003;101(8):2914-2923.

3. Cattoretti G, Pasqualucci L, Ballon G, Tam W, Nandula SV, Shen Q, Mo T, Murty VV, Dalla-Favera R. Deregulated BCL-6 expression recapitulates the pathogenesis of human diffuse large B-cell lymphomas in mice. Cancer Cell 2005;7:445-455.

4. Phan RT and Dalla-Favera R. The BCL6 proto-oncogene suppresses p53 expression in germinal-center B cells. Nature.2004;432(7017):635-639.

5. Phan RT, Saito M, Basso K, Niu H, Dalla-Favera R. Miz-1 mediated suppression of CDKN1A and cell-cycle arrest by BCL6 in germinal-center B cells. Nature Immunol, in press.

Ari M. Melnick, MD, Albert Einstein College of Medicine, Bronx, NY
Targeting BCL-6 in Germinal Center B-Cell Lymphomas

The BCL6 transcriptional repressor is required for establishment of the germinal center phase of differentiation by B cells. BCL6 is often expressed in germinal center derived B-cell lymphomas due to translocations or point mutations that alter regulatory elements in the BCL6 promoter. Our research is focused on the transcriptional mechanism of action of BCL6 and the downstream target genes required for germinal center formation and lymphomagenesis. We have identified several co-repressor complexes that bind to BCL6 and that are required for repression of specific subsets of its target genes. For example, we found that the SMRT, N-CoR, and BCoR co-repressors all bind to a motif in the BCL6 N-terminal domain that is unique and specific to BCL6. To determine the contribution of these three co-repressors to the transcriptional and biological effects of BCL6, we designed a specific peptide-based inhibitor (called BPI). BPI blocked formation of BCL6 repression complexes on specific target genes, reactivated expression of these genes, and abrogated germinal center formation in vivo, similar to the phenotype of BCL6 null animals. To determine whether B-cell lymphomas are dependent on BCL6 expression, we exposed human cell lines and tumors to BPI. Exposure to BPI killed BCL6 positive but not BCL6 negative cell lines and primary tumors both in vitro and in vivo. BPI is currently under development for use in clinical trials for patients with B-cell lymphomas. In contrast, BPI did not induce differentiation in B cells. This was surprising since BCL6 is known to block post-germinal center maturation. Downregulation of BCL6 allows cells to differentiate to plasma or memory cells. However, others and we recently found that BCL6 also recruits the NuRD corepressor complex, which was required for the differentiation blocking effects of BCL6 but not for survival of lymphoma cells. In order to map the target genes of BCL6 required for germinal center formation, differentiation block, and lymphomagenesis, and which co-repressors mediate these effects, we combined several high throughput genomic techniques including “ChIP on chip” and expression arrays. Using BPI and RNAi of different components, we were able to identify the cohort of target genes involved in these processes and determine the mechanism through which they are silenced. Overall, our data indicate that disrupting specific transcriptional repression complexes in normal and malignant B cells allows the contribution of gene pathways and gene regulatory mechanisms required for normal and malignant B cells to be identified, and could serve as novel targeted therapy agents for B-cell lymphomas.

Loren D. Walensky, MD, PhD, Dana-Farber Cancer Institute and Children’s Hospital Boston, Harvard Medical School, Boston, MA
The BCL-2 Family as a Rational Therapeutic Target in Germinal Center Lymphomas

The identification of BCL-2 at the chromosomal breakpoint of t(14;18) lymphomas led to the seminal discovery that cell fate is governed by a complex network of interactions among the BCL-2 family of pro-death and anti-death proteins. The t(14:18) translocation brings the bcl-2 gene under the control of immunoglobulin heavy chain regulatory elements, leading to inappropriately elevated levels of the bcl-2 Ig transcript and overexpression of the BCL-2 survival protein. This chromosomal lesion occurs in over 85% of follicular lymphomas (FL) and 20% of diffuse large B-cell lymphomas (DLBCL), and enables lymphoma cells to evade programmed cell death or “apoptosis”. Deregulation of apoptotic pathways has since been observed in a variety of lymphomas, even in the absence of such chromosomal aberrations, and has been linked to tumor progression and acquired resistance to chemotherapy-induced cell death. The application of genomic, proteomic, biochemical, and murine transgenic technologies to investigate the molecular etiologies of germinal center lymphomas has implicated distinct BCL-2 family members in lymphomagenesis. Although treatment options for lymphoma have expanded in recent years, FL remains incurable and DLBCL is cured in less than 50% of patients. Not surprisingly, apoptotic proteins have become key targets for anti-lymphoma drug development. Novel small molecule, peptidomimetic, peptide-stapling, and antisense strategies are being employed to selectively neutralize anti-apoptotic proteins in order to reactivate programmed cell death in lymphoma cells. These targeted therapeutics have significant potential to expand the treatment arsenal for germinal center lymphomas.

Epigenetic processes involve the establishment of patterns of gene expression via heritable alterations in the chromatin structure of genes; however, the molecular details of how these processes are regulated are poorly understood. We, therefore, asked the following questions: How is active chromatin established at specific genes in early precursor cells? How is chromatin altered during the stages when cell fates are decided, and can we draw general conclusions about how such decisions are made? How are individual lineages specified and restricted, and last, but not least, which factors regulate these processes and how do they do it? To identify the molecular events taking place during developmentally controlled gene activation and silencing, we are examining the regulation of chromatin structure of myeloid specific genes. A paradigm for such a gene is the macrophage-colony-stimulating factor receptor (csf1 or c-fms) gene. This gene is expressed in hematopoietic stem cells (HSCs), is up-regulated in macrophages, and is silenced in lymphoid cells. We have previously shown that the c-fms locus is organized into active chromatin in HSCs 1. Here we show that transcription factor complex assembly and active chromatin formation at the c-fms locus are critically dependent on the presence of the transcription factor PU.1. Using precursor cells from PU.1 knock-out mice carrying an inducible PU.1 gene, we examined how active chromatin structure in precursor cells is established and defined a precise order of assembly events. We have previously shown that c-fms is expressed in restricted lymphoid as well as myeloid progenitor cells, and that it displays a similar chromatin structure, indicating that these cells are epigenetically related¹. To study lineage specification at the chromatin level, we examined silencing of c-fms during B-cell development. We showed that epigenetic silencing of the c-fms locus during B lymphopoiesis occurs in a distinct order and that even mature B cells display a poised chromatin structure. This poised structure correlates with the ability of c-fms to be re-expressed after the conditional inactivation of the B-cell specific transcription factor Pax5. Our experiments indicate that Pax5 has to be present throughout B lymphopoiesis to counteract active chromatin formation and to maintain myeloid-specific genes in a silent state. To gain insight into the molecular mechanism by which Pax5 represses c-fms expression, we have examined how Pax5 alters c-fms chromatin modification and chromatin accessibility. We defined the c-fms promoter as the major cis-regulatory target of Pax5 and the transcription complexes mediating the Pax5 response. Our experiments support a model by which gene expression is primed in HSCs by the selective activation of specific cis-elements. These elements are subject to regulation by cross-regulatory transcription factors leading to lineage-specific activation or silencing.

Reference:

1. Tagoh H, Schebesta A, Wilson N, Hume, DA, Busslinger M, Bonifer C. Epigenetic silencing of the c-fms locus during B-lymphopoiesis occurs in discrete steps and is reversible. EMBO J 2004;23: 4275-4285.

Co-Authors: Nicola Wilson, Hanna Krysinska, PhD, Maarten Hoogenkamp, PhD, Richard Ingram, PhD, and Hiromi Tagoh, PhD, St James’s University Hospital, University of Leeds, Leeds, United Kingdom; Meinrad Busslinger, PhD, Institute for Molecular Pathology, Vienna, Austria; Harinder Singh and Peter Laslo, PhD, University of Chicago, Howard Hughes Medical Institute, Chicago, IL

Emery Bresnick, PhD, University of Wisconsin Medical School, Madison, WI
GATA Factor Interplay at Chromatin Domains

The hematopoietic GATA factors, GATA-1-3, function through simple cis-elements (GATA motifs) to initiate genetic programs that drive hematopoiesis. Whereas GATA factors bind with high affinity to GATA motifs within naked DNA templates, our analyses of GATA-1 and GATA-2 occupancy within broad chromosomal regions in cells revealed occupancy at only a small subset of conserved GATA motifs. Occupancy was not predictable based on GATA motif conservation or the existence of a GATA motif within a DNaseI hypersensitive site. Thus, the rules that govern GATA factor chromatin occupancy are poorly understood. We hypothesize that occupancy is determined by a “GATA Recognition Code” (GRC), in which intrinsic features of the GATA motif, nearest-neighbor cis-elements (and bound factors), chromatin environment, and sequence conservation are critical parameters. To elucidate the GRC, we are using quantitative ChIP, ChIP coupled with genomic microarrays, and computational strategies to analyze GATA-1 and GATA-2 occupancy at endogenous loci in cultured cells and primary hematopoietic precursors. GATA-1 displaces GATA-2 from chromatin target sites, inducing a “GATA switch”. GATA switches instigate altered transcriptional outputs at multiple target genes, including GATA-2, thereby regulating hematopoiesis. GATA switches require Friend of GATA-1, a GATA-1 coregulator that mediates activation and repression in a context-dependent manner. Proteasome-mediated destruction of GATA-2 allows GATA switches to proceed, whereas proteasome inhibition stabilizes GATA-2 and attenuates GATA switches. Thus, GATA-2, FOG-1, and the proteasome are determinants of GATA-1 chromatin occupancy. Unraveling the GRC will yield fundamental insights into physiological mechanisms that regulate hematopoiesis and how alterations in GRC parameters impact upon, and potentially derail, GATA factor function.

Pier Paolo Pandolfi, MD, PhD, Memorial Sloan-Kettering Cancer Center, New York, NY
Aberrant Chromatin Remodeling in Oncogenesis and its Therapeutic Implications: The POKEMON Paradigm

Aberrant transcriptional repression through chromatin remodeling and histone deacetylation has been postulated to represent a driving force underlying leukemogenesis, as histone deacetylase inhibitors have been found to be effective in cancer treatment. However, the molecular mechanisms by which transcriptional derepression would be linked to tumor suppression are poorly understood. We have recently identified the transcriptional repressor POKEMON (encoded by the Zbtb7 gene) as a critical factor in oncogenesis. We found that cells lacking POKEMON are refractory to oncogene mediated cellular transformation. Conversely, POKEMON overexpression overcomes oncogene induced premature senescence and apoptosis, leading to overt oncogenic transformation both in vitro and in vivo in the hematopoietic compartment of transgenic mice. We identified POKEMON as a potent transcriptional repressor of the important tumor suppressor gene ARF through direct binding to its promoter. Furthermore, analysis of complete and conditional KO mutants indicated that the POKEMON proto-oncogene acts as a master regulator of both fetal and adult myeloid and lymphoid hematopoiesis. We will discuss the implications of these findings for the pathogenesis of hemopoietic malignancies, as POKEMON is found aberrantly overexpressed in both leukemia and lymphoma as well as in solid tumors, and its expression levels predict biological behavior and clinical outcome. Taken together, our findings provide a further rationale for transcription-based therapeutic modalities and identify POKEMON as an important target for therapy on the basis of its key role in oncogenesis.

Drosophila mounts a potent host defense when challenged by various microorganisms. The analysis of this defense by molecular genetics has provided a global picture of the mechanisms by which this insect senses infection, discriminates between various classes of microorganisms, and induces the production of effector molecules, among which antimicrobial peptides are prominent. An unexpected result of these studies was the discovery that most of the genes involved in Drosophila host defense signaling are homologous or very similar to genes implicated in mammalian innate immune defenses. In contrast to vertebrates, Drosophila Pattern Recognition Receptors that detect infections belong to two families, the Peptidoglycan Recognition Proteins (PGRPs) and the ß-Glucan Recognition Proteins (also known as GNBPs). Our recent findings indicate that fruitflies use a second strategy to sense infections: the antifungal response can be triggered by fungal virulence factors that are detected through their catalytic activity. We suggest that this dual sensing strategy, the detection of cell wall compounds through PRRs on the one hand, and the perception of virulence factor activity on the other hand, is similar to that used by the innate immune system of plants, and possibly that of vertebrates.

Luke O’Neill, PhD, Trinity College Dublin, Dublin, Ireland
Toll-Like Receptor Signal Transduction During Infection and Inflammation

Toll-like receptors have emerged as key initiators of host defense against pathogens. They recognize a large number of microbial products, providing a repertoire that allows the host to respond to all invading pathogens. Once they are activated, they trigger signaling pathways that culminate in the increased expression of a large number of immune and inflammatory genes. The best characterized TLRs are TLR4, which recognizes LPS, and TLR3, which responds to viral double-stranded RNA. Signaling is driven by the Toll-IL-1 receptor (TIR) domain, which occurs in all TLRs and also in the four adapter proteins implicated in receptor-proximal signaling MyD88, Mal, Trif, and Tram. These adapters are probably recruited to receptor TIR domains, which form a dimeric platform allowing assembly of the initial signaling complex. The adapters then recruit downstream effectors, which include several kinases, notably the IRAKs, RIPs, and TBK-1. Signaling specificity is becoming apparent with TLR3 recruiting Trif, which in turn activates RIP1 and TBK-1, leading to activation of IRF-3. Most other TLRs recruit MyD88, which activates IRAK-4 and RIP2, leading to activation of NF-kappaB and induction of multiple inflammatory genes. This allows for tailoring of the response to invading pathogens and provides specificity to innate immunity, a feature that is becoming increasingly apparent. Specific roles for Mal and Tram have yet to emerge, although there are clear biochemical differences between these and the other adapters. Tram is myristoylated, and recently, we have found that Mal is a substrate for caspase-1 and required cleavage in order to be activated. Endogenous inhibitors of TLRs have also been described which function by sequestering adapters from the signaling pathways, a notable example being ST2. Clearly, we have much to learn about the component parts in TLR signaling and their regulation.

Douglas Golenbock, MD, University of Massachusetts Medical School, Worcester, MA
The Toll-Like Receptor Paradigm: Responses to Bacterial Endotoxin

Toll-like receptors function to recognize the presence of microbial products and to activate many innate immune host defenses. Bacterial endotoxin, or lipopolysaccharide (LPS), constitutes the major proinflammatory molecule on the outer leaflet of the Gram-negative bacterial cell wall. LPS activity is enhanced a thousand-fold by the combined action of LBP and CD14, but neither of these molecules is absolutely essential for LPS stimulatory activity. In contrast, the cell surface molecule, TLR4 and its co-receptor, MD-2, are both required for most, if not all, responses to endotoxin. MD-2 appears to be the major LPS binding component of the endotoxin receptor. Endotoxin antagonists, such as B1287, appear to inhibit the activity of TLR4 by blocking the binding of LPS to MD-2. Once TLR4 is activated, LPS signal transduction requires the adapter functions of four TIR domain containing adapter molecules: MyD88, Mal/TIRAP, TRIF, and TRAM. TRAM is uniquely specific for TLR4 signal transduction. TRAM is a surface-localized protein that depends, in part, on myristoylation at its N-terminus for surface localization. TRAM mutants that lack a N-terminal myristoylation consensus sequence not only fail to co-localize at the surface of cells (and in the Golgi) with TLR4, but are incapable of complementing the LPS-insensitive phenotype of macrophages from TRAM knockout mice. In summary, the ability of mammalian cells to respond to LPS depends on the complex interaction of a family of molecules including LBP, CD14, TLR4, MD-2, and the TIR domain containing adapters.

Co-Authors: Kate Fitzgerald, PhD, Brian Monks, Daniel Rowe, and Alberto Visintin, PhD, University of Massachusetts Medical School, Worcester, MA

Many of the most scientifically validated protein targets for molecular intervention in cancer therapy lack structural features that enable them to be targeted by small-molecule therapeutics. Work in our laboratories has led to the development of several platform technologies that aim to target the “untargetable” through novel synthesis-based approaches. For example, in collaboration with Stanley Korsmeyer’s laboratory, we have demonstrated the ability to interfere with intracellular protein-protein interactions using hydrocarbon-stapled a -helical peptides. We have also shown, in collaboration with Affymetrix Corporation, that folded RNA motifs in cells can be specifically targeted by short RNA-interacting polynucleotides (RIPtides) discovered using microarray screening. Progress on these fronts, with particular emphasis on hematology-related targets, will be reviewed.

Reference:

1. Walensky LD, Kung AL, Escher I, Malia T, Barbuto S, Wright R, Wagner G, Verdine GL, Korsmeyer SJ. Activation of apoptosis by a hydrocarbon-stapled BH3 helix. Science 2004;305:1466-1470.

Co-Authors: Loren D. Walensky, MD, PhD, Dana-Farber Cancer Institute and Children’s Hospital Boston, Harvard Medical School, Boston, MA; and Federico Bernal, PhD, Webster D. Santos, PhD, and Lourdes Gude, PhD, Harvard University, Cambridge, MA

John Kuriyan, PhD, University of California – Berkeley, Howard Hughes Medical Institute, Berkeley, CA (Saturday only)
Conformational Inter-Conversion in the Abl Kinase Domain

The remarkable success of the cancer drug imatinib (Gleevec^®, Glivec^®, STI-571 [Novartis]) stems in part from its ability to specifically block cell proliferation by inhibiting the kinase activity of BCR-Abl, a constitutively activated mutant form of the Abl tyrosine kinase that results from a chromosomal translocation present in the cells of patients diagnosed with chronic myelogenous leukemia (CML). Despite the generally positive response of CML patients to treatment with imatinib, a serious problem that emerges with time is the accumulation of mutations in BCR-Abl that lead to resistance to the drug. Understanding the determinants of inhibitor binding to the Abl tyrosine kinase and to other important kinase targets of cancer therapy, therefore, remains a pressing problem. A surprising aspect of the selectivity profile of imatinib is that the drug is not an effective inhibitor of the closely related Src family tyrosine kinases, c-Src, c-Fgr, and c-Lyn, although it inactivates the more distantly related Kit and the platelet-derived growth factor receptor (PDGFR) tyrosine kinases. Like Abl, the Src kinases are non-receptor tyrosine kinases that contain SH2 and SH3 domains and are located near each other on the kinase evolutionary tree, representing their divergence from a common branch point separate from that of the receptor tyrosine kinases Kit and PDGFR. The kinase domain of Abl is 47% identical in sequence to that of c-Src, whereas it shares only 35% and 36% sequence identity with the Kit and PDGFR kinase domains, respectively. We are, therefore, examining in detail the differences that characterize the inactive states of the Src and Abl tyrosine kinase domains. We have determined the crystal structure of a substrate complex of Abl in which the kinase unexpectedly adopts an inactive conformation. Two features of this structure are of particular interest. First, the activation loop adopts a conformation in which the activation loop tyrosine residue is presented for phosphorylation by another kinase molecule. Second, the conformation of the kinase is strikingly similar to that of inactive c-Src, indicating that Src and Abl can adopt very similar inactive conformations. Detailed analysis of these structures by computer simulation suggests a possible mechanism by which the DFG motif, a central regulatory region of the kinase, undergoes the conformational change required for the kinase to activate. Taken together, these results suggest that these different kinases may pass through similar inactive conformations as intermediates on the pathway between fully active and least active states.

Co-Authors: Nick Levinson, Olga Kuchment, Matthew A. Young, PhD, Markus Seeliger, PhD, M. Nidanie Henderson, PhD, Bhushan Nagar, PhD, University of California - Berkeley, Berkeley, CA, and Kui Shen, PhD, and Philip A. Cole, MD, PhD, Johns Hopkins School of Medicine, Baltimore, MD

Nikola Pavletich, PhD, Memorial Sloan-Kettering Cancer Center, Howard Hughes Medical Institute, New York, NY (Sunday only)
BRCA2 Tumor Suppressor Function in the Repair of DNA Double-Strand Breaks

Inherited mutations in BRCA2 cause an autosomal dominant predisposition to breast and ovarian cancer. BRCA2 is required for the error-free repair of DNA double-strand breaks (DSBs) by homologous recombination. In this process, the RAD51 recombinase forms a nucleoprotein filament with the single-stranded DNA (ssDNA) of the resected DSB, and then catalyzes strand exchange with homologous DNA. The resulting strand-invasion intermediate primes DNA synthesis, copying the missing information from the donor DNA. Loss of BRCA2 reduces the efficiency of homologous recombination-mediated repair and results in genomic instability. Previous studies showed that BRCA2 binds to RAD51 and to ssDNA, with preference for ssDNA-dsDNA junctions. These findings led to the model that BRCA2 targets RAD51 to resected DSBs. To test this model, we established an in vitro recombination assay reconstituted with purified proteins from the fungus Ustilago maydis. Using this model system, we find that BRCA2 stimulates recombination by facilitating the nucleation of the RAD51-ssDNA nucleoprotein filament, known to be a rate-limiting step. BRCA2-nucleated filaments start preferentially at the dsDNA-ssDNA junction, with strict specificity for the polarity of a resected DSB. These results indicate that BRCA2 is needed to initiate the RAD51-mediated repair of DSBs, and highlights the dsDNA-ssDNA junction of the processed DSB as a likely source of repair signaling.

Co-Authors: Haijuan Yang, PhD, Memorial Sloan-Kettering Cancer Center, New York, NY, and William K. Holloman, PhD, Cornell University Weill Medical College, New York, NY

Charles L. Sawyers, MD, University of California – Los Angeles, School of Medicine, Los Angeles, CA
Overcoming Resistance to Kinase Inhibitors

Current evidence suggests a role for Notch signaling in regulating early hematopoietic development. The clearest evidence for a physiologic role of Notch signaling has come from gain-of-function and loss-of-function studies demonstrating that Notch1-mediated, CSL-dependent signaling promotes T-cell but inhibits B-cell development. Although the physiologic role of Notch signaling in regulating hematopoietic stem cell fate decisions in vivo has been controversial, in vitro studies have shown that enforced expression of the constitutively active intracellular domain of Notch1 in murine hematopoietic stem cells induces inhibition of myeloid differentiation and promotion of early T-cell differentiation, and enhances the generation of marrow-repopulating cells. These studies suggest a potential role for Notch signaling in regulating stem cell growth and differentiation and indicate that compensatory mechanisms may obscure this role in vivo. Similarly, activation of endogenous Notch receptors using an immobilized, engineered form of the Notch ligand Delta1 (Delta1^ext-IgG) in non-mutant murine stem cells also profoundly affected differentiation in a cytokine context-dependent manner, including the promotion of early T-cell differentiation and generation of a multi-log increase in the number of precursors with short-term lymphoid and myeloid-repopulating ability. Furthermore, quantitative differences in ligand density and Notch activation affected early T- and B- cell fate decisions, possibly reflecting the differential activation of Notch target genes. In studies of human cord blood precursor cells, culture with Delta1^ext-IgG led to an approximate 15-fold increase in the number of NOD/SCID repopulating cells and provided more rapid engraftment compared to non-cultured cells. Based on these preclinical studies demonstrating enhanced early engraftment of Delta-cultured cells, we have developed clinically relevant methods for expanding cord blood repopulating cells using Notch ligand that will be assessed for their ability to overcome the delayed engraftment often encountered in patients undergoing cord blood transplantation.

Co-Authors: Colleen Delaney, MD, Mari Dallas, MD, and Barbara Varnum-Finney, PhD, Fred Hutchinson Cancer Research Center, Seattle, WA

Geraldine Weinmaster, PhD, David Geffen School of Medicine at University of California – Los Angeles, Los Angeles, CA
Ligand-Induced Notch Signaling: BIND, PULL, GULP, and RIP

Notch signaling regulates a wide variety of cell fates and cellular processes and relies on contact between cell surface DSL ligands (Delta or Jagged) on one cell with Notch receptors on an adjacent cell. Signaling requires ligand-induced Notch proteolysis to release a biologically active Notch intracellular domain (NICD) that directly regulates Notch target gene expression. Although the generation and transcriptional activity of NICD has been intensively studied, little is known regarding the role and fate of the Notch extracellular domain (NECD) produced in response to ligand binding. Studies have indicated that NECD removal is necessary for Notch proteolysis to occur; however, the mechanism of NECD release is not well defined. NECD may be released by ligand-induced proteolytic shedding, but support for this idea has yet to be reported. Alternatively, NECD may be removed by endocytosis into ligand-expressing cells, through a poorly characterized process of trans-endocytosis. However, evidence for transfer of NECD into DSL ligand-expressing cells has only been demonstrated in Drosophila. To obtain support for NECD trans-endocytosis in activation of Notch signaling in mammalian cells, we have imaged the transfer of NECD into DSL ligand-expressing cells, as well as NICD into the nucleus of interacting Notch1 cells following interactions between Notch1 and DSL ligand cells. Transfer of NECD into DSL ligand cells requires furin processing to produce a functional, heterodimeric Notch1 receptor but does not involve additional Notch1 proteolytic cleavage events that regulate NICD production and downstream signaling. Defects in either general or Delta1-specific endocytosis block activation of Notch1 signaling but do not prevent Delta1-induced receptor clustering or movement of Notch1 towards Delta1 cells. Together, our findings provide new insights into the molecular mechanisms that regulate ligand-induced Notch signal transduction.

Jon C. Aster, MD, PHD, Brigham and Women's Hospital, Boston, MA
NOTCH Signaling in T-ALL: From Bench to Bedside

Recent studies have shown that the majority of human acute T-cell lymphoblastic leukemias and lymphomas (T-ALL) harbor gain-of-function mutations in NOTCH1, a heterodimeric type I transmembrane receptor that is required for normal T-cell development. NOTCH1 signals through a mechanism that relies on intramembranous proteolysis, which releases the intracellular domain of NOTCH1 (ICN1), allowing it to translocate to the nucleus and form a short-lived transcriptional activation complex. Two types of gain-of-function mutations are commonly observed in primary lymphoblasts and established T-ALL cell lines: i) juxtamembranous mutations involving the extracellular NOTCH1 heterodimerization (HD) domain that increase ligand-independent ICN1 production; and ii) mutations in the intracellular portion of NOTCH1 that delete a C-terminal PEST "degron" domain and thereby sustain ICN1 action. Not uncommonly, both types of mutations are found in single NOTCH1 alleles, an alignment that leads to synergistic increases in NOTCH signal strength. Similar NOTCH1 mutations are frequently observed in a number of different murine T-ALL models, and both human and murine T-ALLs often undergo growth arrest upon withdrawal of NOTCH signals. These data, which demonstrate a central role for NOTCH1 in T-ALL, have led to the recent opening of a clinical trial of a NOTCH pathway inhibitor in patients with refractory disease, and raise a number of mechanistic questions. One is whether leukemogenic mutations in the HD domain merely weaken contacts that are important for maintaining the integrity of NOTCH1 heterodimers (thus mimicking one model of normal ligand-mediated NOTCH receptor activation), or act through other, more novel mechanisms. Other emerging areas include the identity and function of the negative regulatory sequences that are impacted by PEST domain deletions, the nature of the primary and secondary targets downstream of NOTCH1 that induce and maintain leukemias, and the interaction of NOTCH1 mutations, alone and in tandem, with other proteins that are commonly dysregulated in T-ALL. Ongoing work that aims to fill these gaps is likely to provide important new insights into physiologic and pathophysiologic NOTCH1 receptor activation, regulation, and function.

Data from monoclonal antibody binding studies, electron microscopy, and the crystal structures of the extracellular domains of αVβ3, alone and in complex with the RGD-mimetic peptide cilengitide, and the αIIbβ3 headpiece in complex with various ligands have provided important new insights into the structure and function of these β3 integrin receptors. Working models have emerged in which inside-out activation leading to ligand binding is initiated by rearrangements in the cytoplasmic and transmembrane domains of the α and β subunits, producing loss of leg-leg and/or leg-headpiece interactions. This, in turn, leads to extension of the bent receptor (“switchblade”) and/or movement of the β3 β-terminal domain away from a region in the βA (I-like) domain that controls ligand binding (“deadbolt”). Comparison of the αVβ3 and αIIbβ3 crystal structures suggests that ligand binding is associated with an ~60 ° swing out motion at the junction of the β3 βA (I-like) and hybrid domains. This dramatic reorganization involves movement of the ADMIDAS cation toward the MIDAS cation, breakage of the β3 Met335 carbonyl bond to the ADMIDAS cation, and movements of the βA (I-like) α1 and α7 helices such that there is an ~4-7Å downward shift of the α7 helix via a one turn hydrophobic ratchet. The resulting ~70Å lateral movement of the hybrid domain is thus a prime candidate for initiating outside-in signaling. The specificity of the drugs tirofiban and eptifibatide for αIIbβ3 over αVβ3 is explained by differences in the depth and hydrophobicity of the ligand binding pocket due in part to a unique “cap” subdomain in αIIb and differences in the αIIb and αV residues interacting with the positively charged group in the ligand. In contrast to the extensive knowledge we have about a IIb b 3 structure and function, αIIbβ3 biogenesis is poorly understood. We therefore studied αIIbβ3 biogenesis in cell lines transfected with human αIIb and β3, human megakaryocyte-like cells derived from cord blood cultured with thrombopoietin, and megakaryocyte-like cells derived from murine bone marrow. Our major findings are that 1) αIIb interacts with the membrane-associated chaperone calnexin via the glycan attached to amino acid N15; since the αIIb headpiece would then be predicted to remain associated with the membrane until released from the calnexin cycle, this may explain why the receptor adopts a bent conformation, and 2) αIIb molecules that do not enter into a complex with β3 are retrotranslocated, ubiquitinated, and degraded by a proteosomal mechanism. The new models αIIbβ3 biogenesis, structure, and dynamics provide refined insights into receptor function, and suggest new targets for improved anti-αIIbβ3 therapeutic agents that may avoid the conformational changes, and thus the potential for receptor activation, induced by agents that bind directly to the ligand binding pocket.

Co-Authors: Beau Mitchell, MD, and Deborah L. French, PhD, Mount Sinai School of Medicine, New York, NY; Jihong Li, MD, The Rockefeller University, New York, NY; Tsan Xiao, PhD, Junichi Takagi, PhD, and Timothy A. Springer, PhD, Harvard University, Boston, MA

Sanford J. Shattil, MD, University of California – San Diego, La Jolla, CA
New Frontiers in Platelet Integrin Signaling

Integrin αIIbβ3 is required for platelet aggregation and spreading during hemostasis and thrombosis. The αIIb and β3 extracellular domains are each fused to a single-pass transmembrane (TM) domain and a short cytoplasmic domain devoid of catalytic activity. αIIbβ3 adhesive functions require binding of fibrinogen or von Willebrand factor to the extracellular domains. This process is regulated by agonist-dependent “inside-out” signals that modulate αIIbβ3 conformation (affinity) and oligomerization (valency). Fibrinogen binding further stabilizes the active conformation of αIIbβ3, promotes integrin oligomerization, and triggers “outside-in” signals that reorganize the actin cytoskeleton for efficient thrombus formation. Recent advances in understanding bidirectional αIIbβ3 signaling include: a) analysis of high-resolution structures of αIIbβ3 and αVβ3; b) discovery that talin regulates integrin affinity by binding to the β3 cytoplasmic domain; c) demonstration that binding of adhesive ligands or talin each induce separation of the αIIb TM and cytoplasmic domains from those of β3; and d) discovery that specific intracellular proteins, such as c-Src, integrin-linked kinase, CIB and protein phosphatase 1, may control αIIbβ3 signaling by direct interactions with the αIIb or β3 cytoplasmic domain. However, these insights have led to new questions relevant to αIIbβ3 as well as other integrins. How is talin binding to αIIbβ3 regulated by inside-out signals? How do other signaling molecules implicated in the control of αIIbβ3 activation, such as Rap1b and CIB, function and what are their relationships to talin? What are the structural changes in the TM and cytoplasmic domains that propagate integrin signals? Do some inside-out signals regulate αIIbβ3 affinity while others regulate oligomerization? How does fibrinogen binding to αIIbβ3 trigger activation of integrin-associated enzymes, such as c-Src, to initiate outside-in signaling? How do integrin-proximal events lead to dynamic changes in the actin cytoskeleton? Are defects in αIIbβ3 signaling important causes of platelet dysfunction in vivo? Can molecular targets be identified in αIIbβ3 signaling pathways for therapeutic purposes?

Co-Authors: Ararat Ablooglu, PhD, Charito Buensuceso, PhD, Teresa Helsten, MD, Hideo Hirakata, MD, Ana Kasirer-Friede, PhD, Barry Moran, Manjula Pandey, PhD, Nick Prevost, PhD, Alessandra Soriani, PhD, Naohide Watanabe, MD, and Jun Yamanouchi, MD, University of California – San Diego, La Jolla, CA

Paul F. Bray, MD, Baylor College of Medicine, Houston, TX
Functional and Clinical Consequences of Platelet Polymorphisms

Prior to 1996, the only variations considered in the genes encoding integrin αIIbβ3 (ITGA2B and ITGB3) were loss-of-function mutations. Subsequently, there has been a large number of studies which have tested the hypothesis that these and other platelet genes harbor common gain-of-function, prothrombotic polymorphisms. The Pl^A1/A2 (Leu33Pro) platelet polymorphism on integrin β3 has a typical scientific history: it was first discovered to cause alloimmune thrombocytopenia, then the immunology and molecular biology were elucidated, and later, associations were demonstrated with clinical thrombotic events. This polymorphism is (mostly) unique in having had extensive basic research investigation on the effect of the Leu33Pro change. To overcome the limitation caused by heterogeneity of human platelets, we generated cell lines over-expressing either the Pl^A1 or Pl^A2 form of αIIbβ3, and observed increased adhesion, spreading, migration, and actin cytoskeletal reorganization in the Pl^A2 cells. Increased outside-in signaling to extracellular signal-regulated kinase 2 and myosin light chain in the Pl^A2 cells, perhaps due to a reduced phosphatase activity, may account in part for the increased adhesive phenotype of the Pl^A2 cells. We have reported that Pl^A2-positivity was twice as common in patients with acute coronary syndromes (ACSs) compared to in-patients with similar traditional risk factors but who lacked coronary disease. Subsequent meta-analyses indicate that the Pl^A2 polymorphism presents a small but significant risk for cardiovascular disease. Novel and cutting-edge genetic work in this field has been extended to other platelet genes. Platelet glycoprotein polymorphisms modify the bleeding risk in type 1 VWD, after placement of ventricular assist devices, after treatment with the αIIbβ3 blocker orbofiban, after subarachnoid hemorrhage and in patients with Glanzmann thrombasthenia. State-of-the-art approaches are now being applied to platelet gene polymorphism identification, linkage disequilibrium of these polymorphisms and identification of haplotypes and tag-SNPs. These will be crucial for large scale candidate gene and genomic scan approaches for the identification of novel genes influencing platelet and clinical thrombosis phenotypes. Lastly, inherited variations in platelet genes modify the effects of medications, including aspirin, αIIbβ3 inhibitors, clopidogrel, aurintricarboxylic acid, sex hormones, and statins. We genotyped 2,145 women in the Heart and Estrogen/Progestin Replacement Study followed for ACS events for seven years and found that common combinations of polymorphisms in the genes for GPIbα and GPVI were associated with hormone therapy harm, benefit, or no effect. If corroborated in future studies, platelet genotyping could be used to predict postmenopausal hormone safety.

David A. Wilcox, PhD, Medical College of Wisconsin, Milwaukee, WI
Therapeutic Expression of αIIbβ3 in Murine and Canine Models of Glanzmann Thrombasthenia

While individual inherited platelet disorders are rare, in aggregate, these diseases can account for a bleeding diathesis in ~1:20,000 individuals. One classic disease is Glanzmann thrombasthenia (GT), a rare autosomal-recessive bleeding disorder that is characterized by genetic defects of the platelet-specific integrin, αIIbβ3. Molecular abnormalities in either the αIIb- or β3-subunit can disrupt receptor synthesis, assembly, and/or function thereby preventing platelets from binding the receptors major adhesive ligands (von Willebrand factor and fibrinogen) to form platelet aggregates as a primary response to vascular injury. As a model to develop methods for gene therapy of platelet defects, GT is especially challenging because platelet integrin level as well as genetic transfer efficiency can play a crucial role in restoring normal hemostasis. Developing effective treatment for animal models for this disease should ensure that a similar strategy would be a viable approach for correcting other inherited platelet disorders. In experiments to be presented, bone marrow from integrin β3-deficient mice was transduced with an integrin lentivirus vector encoding human β3 driven by the tissue-specific αIIb promoter. Transduction of this vector into marrow stem cells restored integrin αIIbβ3 to the surface of platelets, which led to improved platelet aggregation and reduced bleeding within this small animal model for GT. As the next step toward a practical approach for human gene therapy, a clincally relevant genetic transfer protocol is currently being examined in a canine model for GT resulting from a defect in integrin αIIb. These studies should help us to define molecular and cellular events necessary for targeting therapeutics to platelets, which will contribute to eventual, safe, effective, and technologically viable strategies for correcting inherited bleeding disorders in humans.

Reference:

1. Fang, J, Hodivala-Dilke, KM, Johnson, BD, Du, LM, Hynes, RO, White II , GC, Wilcox, DA, Therapeutic expression of the platelet-specific integrin, αIIbβ3, in a murine model for glanzmann thrombasthenia, Blood 2005, June 21; [Epub ahead of Print].

Co-Author: Mary K. Boudreaux, DVM, PhD, College of Veterinary Medicine, Auburn University, AL

While ES cells have the potential to give rise to most differentiated cell lineages, in vitro control of their differentiation is still a difficult task requiring extensive efforts from various directions. Such efforts include 1) specification of steps required for differentiation of a given cell, 2) development of markers to define those intermediate steps, and 3) specification of the molecular signaling regulating each step. Moreover, there is increasing expectation that in vitro ES cell differentiation can serve as a model for studying embryonic development at the cellular level. DNA microarrays should be useful tools for these studies. Over the last several years, we have developed surface markers and ES cell lines that allow us to define and purify intermediate stages appearing in ES cell differentiation culture. Those intermediate populations have been collected and subjected to DNA microarray analysis, which has developed into a continuously expanding database of transcription based on Affymetrix oligonucleotide arrays. This database currently contains ca. 100 purified samples, including more than 20 distinct early intermediates that appear during ES cell differentiation, representing all three primary germ layers, and also a similar number of samples derived from embryonic and adult tissues which provide a powerful transcriptional context aiding the meaningful analysis of the differentiation data. In order to process the data generated in this process, we have developed novel analysis systems that allow both analysis and dissemination of the data. This system does not aim to answer narrowly defined questions, but rather facilitates the rapid appraisal of results obtained by fuzzy queries by providing both expression and genomic context in a convenient graphical interface. More important, this system allows the data to be accessed concurrently by a number of researchers specializing in various aspects of ES cell differentiation, thus allowing the biologically relevant information to be extracted. In this session, I will revisit the issue of stemness genes by using this database and would like to point out the importance of analysis software that enables dialogues with a large volume of data sets. Ironically, researchers using microarray analysis, though dealing with enormous complexity, often neglected it. Next, I will show the utility of our database as a platform for functional genomics to understand the biological significance of a particular gene, referring to a gene that is involved in neural crest differentiation and may be implicated in oncogenesis of neuroblastoma. Finally, I will show the utility of this database to identify genes involved in differentiation of endothelial and hematopoietic cell lineages. The value of this database increases when combined with a reliable and rapid functional assay. I will demonstrate our method to reveal the molecular hierarchy in the process of mesendoderm differentiation, which is based upon RNA interference and a highly selective culture condition with a defined medium. In conclusion, DNA microarray technology in combination with ES cell differentiation culture has an enormous potential to dissect the process of cell specification during embryogenesis. The information derived from this approach is able to provide feedback on ES cell differentiation in culture, thereby determining molecular requirements for inducing guided differentiation of ES cells.

Co-Authors: Lars Martin Jakt, PhD, and Takumi Era, MD, PhD, Stem Cell Research Group, Center for Developmental Biology, Kobe, Japan

Gordon Keller, PhD, Mount Sinai School of Medicine, New York, NY
Lineage Specific Differentiation of Embryonic Stem Cells

The ability of embryonic stem (ES) cells to differentiate to a broad spectrum of cell types in culture offers outstanding opportunities to study the molecular events determining lineage specification as well as a source of cells and tissues for transplantation for cell-based therapy. For this potential of the ES cell system to be realized, however, it is essential to understand the processes that lead to the induction of the three primary germ layers, ectoderm, mesoderm, and endoderm. To investigate these early differentiation events, we targeted the green fluorescent protein (GFP) cDNA to the mesodermal gene, brachyury (bry). Using these ES cells (GFP-Bry), it is possible to track the formation of mesoderm and isolate mesodermal cells by sorting GFP ^pos cells from the differentiation cultures. With this model, we have shown that mesoderm that generates hematopoietic and vascular cells is distinct from mesoderm that gives rise to the cardiac lineage. This is an important observation as it demonstrates that cell fates are established early in development. Together with the expected mesoderm derivatives, we have also shown that endoderm derivatives, including hepatocyte-like cells and gut tissue also develop from a brachyury^pos progenitor. These findings indicate that at least a portion of the brachyury^pos population has endoderm potential. To be able to isolate endoderm from the brachyury positive population, we have targeted the human CD4 cDNA to the Foxa2 (HNF3 b) locus in the GFP-bry cells. Using these CD4-Foxa2/GFP-Bry ES cells, we are able to induce a cell population that has characteristics of the primitive streak of the mouse embryo. CD4-Foxa2^posGFP-bry^pos cells that develop in the differentiation cultures show gene expression patterns and potential similar to that of the anterior primitive streak whereas CD4-Foxa2^neg GFP-bry^pos cells display characteristics of the posterior region of the streak. When cultured on matrigel, the CD4-Foxa2^posGFP-bry^pos population generates distinct colonies of cells that express Foxa2, α fetoprotein, and albumin, suggesting that they represent developing hepatocytes. Current experiments are focused on transplanting these hepatocyte-like cells into mouse models of liver failure.

Co-Authors: Paul Gadue, PhD, Valerie Gouon-Evans, PhD, and Mount Sinai School of Medicine, New York, NY

George Q. Daley, MD, PhD, Children's Hospital Boston, Boston, MA
Modeling Hematopoietic Transplantation with Embryonic Stem Cells

Differentiation of embryonic stem (ES) cells in vitro yields abundant hematopoietic progenitors, but achieving stable hematopoietic engraftment of irradiated mice has proven difficult. This has been attributed to the likelihood that ES cell differentiation recapitulates the earliest yolk sac stages of hematopoietic development, when progenitors have limited developmental potential and fail to productively engraft adults. In an effort to characterize factors that promote formation of definitive hematopoietic stem cells (HSCs), we have shown that activation of a genetic pathway involving the homeodomain proteins cdx4 and hoxb4 promotes the differentiation of blood progenitors that engraft stable lymphoid-myeloid hematopoiesis in irradiated mice. The cells provide radioprotection, reconstitute B220/CD19 and B220/IgM positive B lymphoid populations in spleen and lymph nodes, CD3/CD4 and CD3/CD8 positive T cells in spleen, and CD4/CD8 double positive T cells in the thymus, and can be transplanted from primary into secondary mice. Southern hybridization of purified populations of myeloid and lymphoid lineages shows multiple common retroviral integration sites, thereby demonstrating the derivation of hematopoietic stem cells from ES cells. We are currently testing whether engraftable HSCs derive directly from primitive progenitors whose fate has been altered by genetic modification, or represent in vitro expansion of rare definitive progenitors. We have used hoxb4 modification of ES-derived HSCs to enable therapeutic transplantation with products of ES cells, including a proof-of-principle application of therapeutic cloning to treat an immunodeficient Rag2^-/- mouse, and efforts are underway in murine models of additional genetic disorders of the bone marrow. Our studies in the blood forming system are aimed at establishing principles applicable to generating engraftable HSCs from human ES cells.

A cause and effect relationship between thrombosis, thrombin generation, and cancer was first recognized by the observation that experimental pulmonary metastasis could be enhanced with minute concentrations of i.v. thrombin. This has been attributed to the effect of thrombin on platelets as well as tumor cell adhesion, growth, seeding, metastasis, and angiogenesis. Thrombin-activated platelets form platelet tumor aggregates through platelet integrin GPIIb-GPIIIa bridges with fibronectin (Fn), VWF, other ligands and tumor integrins, as well as platelet P-selectin and its receptor on tumor cells. This prolongs the life of tumor cells in the host by preventing their rapid clearance from the circulation by NK cells. Activated platelets also release tumor growth factors (PDGF) and vascular factors (VEGF and Ang-1). Experimental pulmonary metastasis is markedly inhibited in thrombocytopenic mice during the first six hours of tumor implantation. It is also inhibited by anti-platelet GPIIIa Ab in the absence of thrombocytopenia. Most tumor cells have constitutively active Tissue Factor (TF) on their surface capable of generating thrombin. TF is associated with the leading edge of tumors in vivo and tumor TF density correlates with metastasis. Thrombin can also activate tumor cells by binding to its PAR-1 receptor on tumors. PAR-1 density correlates with metastasis. PAR-1 activated tumor cells enhance their adhesion to platelets, endothelial cells, Fn, and VWF. In vivo tumor cell implantation, growth, seeding, and spontaneous metastasis are inhibited by the potent specific thrombin inhibitor, hirudin – indicating the elaboration of endogenous thrombin in the platelet-tumor environment. Thrombin can also induce the production and secretion of various vascular growth factors from tumor or endothelial cells (VEGF, KDR, Angiopoietin-2, MMP 1 and 2, and GRO-α). GRO-α upregulation by thrombin is critical for upregulation of other vascular growth factors. Thrombin stimulates endothelial growth as well as endothelial cord formation and enhances in vivo neoangiogenesis. The effects on neoangiogenesis are inhibited by an Ab against GRO-α or in cells in which GRO-α has been knocked down. There is evidence that thrombin activates a dormant tumor state. Patients treated for deep vein thrombosis with coumadin for six months developed less cancer over six years than those treated for six weeks. Hypercoagulable males develop cancer at a greater incidence than others over 11 years of observation. Several clinical studies have demonstrated low-grade DIC in cancer patients which is proportional to tumor burden. Coumadin or LMWH have prolonged life in cancer patients with low tumor burden. Thus, thrombin generation by cancer cells contributes to the induction of a malignant phenotype. It is suggested that this may be due to the activation of a dormant tumor state. A controlling factor in the development or exacerbation of cancer may reflect a balance between pro-thrombotic and anti-thrombotic endogenous regulators in the host.

References:

1. Hu L, Lee M, Campbell W, Perez-Soler R, Karpatkin S. Role of endogenous thrombin in tumor implantation, seeding and spontaneous metastasis. Blood 2004;104:2746-2751.

2. Karpatkin S. Does hypercoagulability awaken dormant tumor cells in the host? J Thromb Haemost 2004;2:2103-2106.

Wolfram Ruf, MD, The Scripps Research Institute, La Jolla, CA
Tissue Factor in Tumor Biology

Tissue Factor (TF) supports three major aspects of tumor biology: primary tumor growth, metastasis, and angiogenesis. In metastasis, TF-dependent thrombin generation triggers platelet- and fibrin-deposition, as well as tumor cell protease activated receptor (PAR) 1-dependent signaling to achieve metastatic arrest. Thrombin pathways synergize to protect tumor cells from host defense or apoptosis and stabilize tumor cell adhesion to the extracellular matrix at sites of metastatic implantation. In contrast, thrombin-independent, direct TF signaling pathways emerged as crucial promoters of angiogenesis and tumor growth. TF serves as the anchor for two distinct signaling complexes: the binary TF-VIIa complex cleaves PAR2 and the coagulation-initiation ternary TF-VIIa-Xa complex activates PAR1 or 2 in dependence of Xa. A regulatory role for the TF cytoplasmic domain in PAR signaling was uncovered in TF cytoplasmic domain deleted mice which exhibit enhanced tumor and developmental angiogenesis. Angiogenesis in TF cytoplasmic domain deleted mice is dependent on PAR2, platelet-derived growth factor BB, and VIIa, but not Xa, indicating synergy of TF-VIIa signaling with a specific pro-angiogenenic growth factor pathway. TF cytoplasmic domain deleted mice may mimic deregulated angiogenesis induced by TF hyperphosphorylation which is specifically detected in pathological neo-vasculature. TF is also expressed by tumor cells, and invasive areas of tumors are known to intensely stain for TF and VIIa. Tumor cell TF-VIIa signaling induces expression of pro-angiogenic mediators and TF promotes primary tumor growth through thrombin-independent pathways. Specific inhibition of TF coagulation or signaling provides evidence that TF promotes metastasis by coagulation activation, whereas tumor growth is driven by TF-VIIa signaling. Thus, efficacy in targeting TF signaling should be considered a necessary component for inhibitory intervention with TF pathways in tumor biology.

Richard Hynes, PhD, Massachusetts Institute of Technology, Howard Hughes Medical Institute, Cambridge, MA
Contributions of Vascular Cell Adhesion to Tumor Progression

The cells of the vascular lining, endothelial cells, and circulating blood cells, platelets and leukocytes, make both positive and negative contributions to tumor progression. These include the roles of endothelial cells in angiogenesis and in adhesion of tumor cells from the circulation during metastasis. There is also abundant evidence for the contributions of platelets to enhancing metastatic spread, and leukocytes can enhance or suppress tumor progression, depending on the particular system (tumor and leukocyte type) under study. In most of these roles, the adhesive properties of the vascular cells play central, but incompletely understood, roles. Key vascular adhesion receptors are selectins and integrins, most particularly the two integrins αvβ3 and αIIβb3. We have been using mice deficient in one or more of these receptors to investigate their contributions to tumor progression, including both primary tumor growth and metastatic spread. Tumor development in mouse tumor models on differing genetic backgrounds lacking specific adhesion receptors assesses the roles of these receptors on both the tumor cells as well as vascular and stromal cells. Using bone marrow transplants and/or transplantable tumors, we can dissect which of these compartments is dependent on a given adhesion receptor. Such experiments implicate selectins recruiting both platelets and leukocytes in enhancing metastatic spread and implicate both selectins and integrins in the innate immune response to transplantable tumors. We are focusing on the possibility that the latter effect is due to their role in recruitment of natural killer cells to the site of the tumor. In other experiments, using an endogenous mammary carcinoma model (MMTVPymT), we detect no role for β3 or β5 integrins either on the tumor cells themselves, or on other cells in the animal, in promoting or suppressing primary tumor growth or metastatic spread. These latter results are somewhat surprising, given earlier reports of positive contributions of αIIβb3 on platelets to metastatic spread. αIIβb3 is also essential for thrombosis, and our results show that its absence does not compromise metastatic spread in the endogenous mouse model studied. Thus the contributions of platelets and thrombosis to the progression of different tumor models clearly differ. The same is true for the roles of αvβ3 and αvβ5 in tumor angiogenesis – they appear never to be essential, but in some cases and not others, they are negative regulators of tumor angiogenesis.

It has become clear that adult mammalian bone marrow contains not one but two ostensibly discrete populations of adult stem cells. The first, and by far the most fully characterized, are the hematopoietic stem cells (HSC) responsible for maintaining lifelong production of blood cells. The biological characteristics and properties of the second marrow resident population of stem cells, variously termed bone marrow stromal cells or mesenchymal stem cells (MSC), are in contrast much less well understood. In vitro, cultures established from single-cell suspensions of bone marrow from a wide range of mammalian species generate colonies of adherent marrow stromal cells, each derived from a single precursor cell termed a colony-forming unit-fibroblast (CFU-F). Culture conditions have been developed to expand MSC in vitro while maintaining the capacity of these cells to differentiate into bone, fat, and cartilage. There is considerable interest in the use of MSC as a cellular therapy for the treatment of not only musculoskeletal defects and diseases, but also in a range of other clinical applications, such as cardiovascular repair. Of particular note are recent data indicating the unique immunosuppressive properties of MSC, which suggest that these cells may find application in a range of transplant settings. The fact that relatively little is known about the precise phenotypic characteristics of the primary clonogenic stromal precursors in the bone marrow responsible for initiating MSC growth in vitro coupled with their low incidence in the marrow has meant that much of our current knowledge of MSC has been gained through in vitro assays and culture manipulations. In defining MSC by their in vitro properties, much consequently remains unknown about the cellular identity, ontogeny, and anatomical location of MSC in the marrow in vivo. In addition, a physiological role for MSC has not been established. Seeking to address these issues, we have sought to develop methodologies to prospectively isolate MSC in highly enriched form from primary hematopoietic tissues in order to explore the biological properties of these cells in an unmanipulated state, unaltered by culture epiphenomena. For the isolation of MSC from human bone